The brain microvasculature is comprised of a specialized class of endothelium that forms a cellular barrier between the bloodstream and the interstices of the brain. This so-called blood-brain barrier (BBB) distinguishes the brain microvasculature from peripheral vascular beds because it constitutes a physical and metabolic barrier that tightly regulates brain uptake of ions, small molecules, proteins, and circulating cells. Since brain endothelial cell plasma membranes contact both the bloodstream and brain interstitial fluid, they are ideally positioned to act as the controlling interfaces for signaling, immune regulation, and transport between the blood and brain. Therefore, many of the unique characteristics of the BBB endothelium can likely be attributed to the protein composition of its plasma membranes. Comprehensive differential plasma membrane protein profiling is necessary to identify these phenotypic determinants, but technological limitations have restricted the global analysis of these comparatively insoluble proteins. To date, studies of BBB membrane protein content are lacking and our understanding of this dynamic interface continues to be governed by a limited number of known physiologic and biochemical attributes. Therefore a novel methodology, subtractive antibody expression cloning, will be used to identify differentially expressed BBB plasma membrane proteins compared with those that are expressed in the highly vascularized liver, kidney, lung, and heart tissues. This antibody-based method will be expanded by using combinatorial human single- chain antibody (scFv) libraries to interrogate the plasma membrane surface of intact brain endothelial cells. This complementary method will result in the simultaneous identification of BBB-specific plasma membrane proteins and cognate scFv targeting reagents. The tissue distributions of the identified BBB-specific plasma membrane proteins will be evaluated in order to determine the quantitative differences in plasma membrane protein expression that exist between the BBB and the aforementioned peripheral vascular beds. The compilation of the differential BBB membrane proteome will help elucidate unique aspects of the BBB tight junction composition, the BBB molecular transport network, and the BBB capacity for participation in disease pathogenesis. Finally, identified BBB-specific plasma membrane protein-scFv pairs will be investigated for their potential as noninvasive drug delivery conduits.